Confirmation of ethanol administration in racing greyhounds by LC-MS-MS.

Ethanol is a prohibited substance in professional animal racing as its administration causes physiological effects such as depression of the central nervous system. Regulation of potential doping agents, including those that inhibit performance, is critical to ensure integrity and animal welfare in greyhound racing, but the detection of ethanol is complicated by dietary and/or environmental exposure. In response, a reliable analytical method capable of detecting recent ethanol administration in greyhound urine samples was validated and implemented. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) was used to investigate the variation in urinary ethanol metabolites; ethyl-ß-D glucuronide (EG; γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ = 1.0 µg/ml, s EG $$ {s}_{\mathrm{EG}} $$ = 3.3 µg/ml) and ethyl sulfate (ES; γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ = 0.9 µg/ml, s ES $$ {s}_{\mathrm{ES}} $$ = 1.9 µg/ml) levels from a reference population of 202 racing greyhounds. These were compared to urine samples collected following administration of ethanol to one male and one female greyhound. Results were used to establish a threshold within the national rules of greyhound racing: γ ¯ EG $$ {\overline{\gamma}}_{\mathrm{EG}} $$ and γ ¯ ES $$ {\overline{\gamma}}_{\mathrm{ES}} $$ > 20 µg/ml in urine are defensible criteria to confirm ethanol administration to greyhounds. Case studies of competition samples are provided to demonstrate the forensic translation of this work.

Biomarker ratios.

The concept of biomarker measurements in the form of a ratio has not been explored in detail. This is surprising considering the current and future potential for biomarkers incorporating endogenous reference compounds (ERCs) in a range of fields. A selection of these relating to clinical and forensic applications, human antidoping, equine antidoping and veterinary residues are discussed.

Testing for Prohibited Substances in Rural Agricultural Show Exhibits: A Twenty-Three Year Experience.

The administration of prohibited substances has been used in agricultural show competitions and animal racing industries to gain unfair competitive advantages. We report the first large prospectively designed descriptive study of drug testing in four species (n = 1,598) over a 23 year period. 4.7% of tested exhibits returned positive results. Commonly detected substances included legitimate veterinary therapeutics such as the sedative acepromazine and the non-steroidal anti-inflammatory phenylbutazone. Targeted testing was more likely to return a positive result than random screening (50 vs 4.7% respectively) although numbers in this targeted sample were small (n = 12). Random drug testing programs were successful in detecting the minority of exhibits using prohibited substances although a wide variety of drugs were found to be used. Further vigilance and research is required in an ever-changing competitive climate to remain at the forefront of detecting new medications in animal show competitions.

Intelligence-based anti-doping from an equine biological passport.

The move towards personalized medicine derived from individually focused clinical chemistry measurements has been translated by the human anti-doping movement over the past decade into developing the athlete biological passport. There is considerable potential for animal sports to adapt this model to facilitate an intelligence-based anti-doping system. Copyright © 2017 John Wiley & Sons, Ltd.

Doping control study of AICAR in post-race urine and plasma samples from horses.

Acadesine, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.